Our recognition of the fundamental mechanisms mRNA expression, exemplified by the regulated expression of HIV, provided the basics for our interest in developing preventive HIV DNA based vaccine strategies. Attractive features of the DNA platform lie in its simplicity, versatility, stability, with repeated administration without vector immunity, being a non-replicating vaccine and not association with adverse effects. Several DNA vaccines are currently in clinical trials against HIV and cancer, and there is a licensed DNA vaccine against dog melanoma. We are testing immunogenicity of SIV and HIV DNA vaccines in mice and selected DNA candidates advance to the macaque model. Some of our successful candidates have been moved to clinical trials. Because intramuscular injection of DNA induces relatively low immune responses in macaques and humans, we are testing additional delivery methods, including in vivo electroporation and liposomes. We reported that electroporation dramatically increased the efficiency of DNA delivery in naive macaques, leading to greatly augmented antigen expression and resulting in the induction of highest levels of T cell responses. Our DNA vaccine includes the cytokine IL-12 DNA (expressed from optimized DNA) as adjuvant and we reported increased magnitude and quality of the responses. Importantly, we reported the dissemination of the DNA vaccine induced T cell responses to mucosal sites including rectal and vaginal mucosa, the portal of entry of HIV. We also found that DNA induced immune responses show extraordinary longevity in vaccinated macaques detectable for several years after the last vaccination. DNA vaccination elicits moderate humoral immune responses in macaques. Using DNA-only vaccination, we found that our optimized DNA vaccine vectors are able to induce potent immune responses able to protect from high viremia. We showed that a protein boost can induce higher levels of Ab. We found that co-delivery of DNA+protein, using either unadjuvanted or adjuvanted protein, in the same muscle at the same time increased antibody production and mucosal dissemination. DNA+Protein co-immunization is superior to vaccination with either of the two individual components in eliciting humoral immune responses. Combination of DNA+Protein induces potent humoral responses able to significantly delay or prevent virus acquisition and improve virological control of the highly pathogenic SIV challenge. To optimize vaccine-induced immunity and efficacy of the DNA+protein co-immunization vaccine regimen, a SIVmac251 based vaccine was compared using two TLR-4-based liposomal formulations as adjuvants (TLR-4+TLR-7 or TLR-4+QS21) in macaques, which resulted in induction of robust humoral immune responses with qualitative differences. Vaccinees were preferentially infected by SIVsmE660 transmitted founder T/F virus carrying the neutralization resistant A/K mutation, demonstrating a strong vaccine-induced sieve effect. SIVsmE660-specific systemic neutralizing antibody and mucosal antibodies targeting V2 region of Env correlated with delay in acquisition of the neutralization-sensitive virus and. V2-specific antibodies and cellular SIV-specific T cell responses contributed to control of viremia. Although both DNA+protein vaccine groups show delay of virus acquisition, the TLR4+7 adjuvanted vaccine induced stronger protective responses resulting in lower peak and chronic viremia. We are further exploring approaches to improve immunogenicity targeting the V2 epitopes to induce more effective responses. We also compared immunogenicity and protective efficacy of a DNA+Protein vaccine delivered following two different strategies: co-administration in the same site or separate delivery of DNA and protein in opposite anatomical sites. We found that the co-immunization induces higher vaccine-induced Env Ab and T cell responses. Importantly, only the co-immunization group showed significant delay in SHIV acquisition with a 67% reduction in per exposure acquisition risk relative to the controls after 15 weekly intravaginal challenges. These data indicate that simultaneous recognition of the two vaccine components (DNA and protein) by the draining lymph node plays a critical role in the development of protective immunity. We are identifying correlates of protection from disease development, which will provide critical information to further improve our vaccine approaches. The advantage of co-immunization vaccine regimens targeting immunogens to the same draining lymph node could be applicable to other vaccine modalities and other pathogens.